The regulation of the initiation of translation is studied on endogenous polyribosomes in crude extracts from HeLa cells. Cell response to protein synthesis inhibition enhances in vitro initiation rates. A controlling and a polyribosome associated actinomycin sensitive factor have been detected. 5-Azacytidine poisons the polyribosome factor. Forty percent of HeLa MRNA is poly(A) minus and has different sequences than poly(A) plus mRNA by cDNA hybridization. The kinetics, drug-sensitivity, turnover and cell localisation are being studied. A particulate HnRNA subfraction contains 10 percent of nucleoplasm RNA but 70 percent of chasable nuclear poly(A). The origin of mRNA in this fraction will be studied. The poly(A) segment of membrane-bound polyribosome mRNA is attached to membranes, half by a "loose" and half by a "tight" attachment. The nature of the poly(A) attachment will be studied. cDNA hybridization will be used to study the "tight", "loose" and "free" mRNA. RNA and protein metabolism regulation in relation to serum controlled growth is studied in 3T6 monolayers and CHO suspension culture. Resting cultures are stimulated into synchronous growth by the addition of 10 percent serum. Ribosomal RNA synthesis increases 5-fold due either to gene activation or increased gene utilization. The Miller spreading technique will be used on nucleoli to determine mechanism. Messenger RNA production rises three-to-five-fold with no increase in HnRN transcription or nuclear poly(A) formation. Messenger production is regulated at the post-transcriptional level, and this mechanism will be further investigated. Messenger RNA content is important in protein synthesis control in serum stimulated cells but the relative importance of initiation control will be measured. The formation of both large HnRNA and the small particulate subfraction will be studied in resting and growth stimulated cells. Comparative studies on invertebrate cell macromolecular metabolism will be continued. The study of selective inhibitors of RNA metabolism will be extended to their effect on growth regulating processes.